The molecular mechanisms that control synapse formation and function on neurons are not known. To gain insights into these mechanisms, the proposed studies will focus on a specific component of nicotinic cholinergic synapses, the neuronal nicotinic acetylcholine receptor (AChR). In particular, the role of innervation in regulating AChR gene expression and AChR function, number and distribution will be investigated in developing chick parasympathetic ciliary ganglion neurons during the process of synaptogenesis. For these studies, the sole source of presynaptic inputs to the ganglion, the accessory oculomotor nucleus, will be surgically ablated at a time in development which precedes synapse formation. To determine whether innervation induces AChR gene expression, precursor and mature AChR mRNA levels will be compared in ciliary ganglion neurons developing in situ in the presence and absence of preganglionic tissue interactions using RNase protection assays and in situ hybridization. The effects of innervation on AChR function will be determined by examining the whole-cell AChR response and the relative abundance and kinetics of the different AChR subtypes on input-deprived and control neurons using whole-cell and single-channel recording methods. The total number of surface AChRs (including silent receptors) on the neurons will be established using a radio-labeled anti-AChR mAb. The role of innervation in the regulation of AChR distribution on the neuron surface will be investigated by indirect immunofluorescence labeling with the anti-AChR mAb. If AChR clusters are observed on neurons developing in situ when innervation is surgically prevented, ultrastructural techniques will be used to determine whether other synaptic specializations, such as the postsynaptic density, are assembled and maintained at these sites. To examine the ability of neurally-derived factors to regulate neuronal AChR and AChR mRNA levels, ciliary ganglion neurons, after developing in the presence and absence of innervation in situ, will be grown in culture in the presence of the appropriate preganglionic tissue, or media conditioned by preganglionic cells. In addition, specific neurally-derived factors that regulate AChR and AChR mRNA levels in muscle, such as AChR-inducing activity (ARIA), calcitonin gene-related peptide and ascorbic acid, will be tested for their ability to influence AChR and AChR transcript levels in neurons. These studies will identify some of the signals and molecular mechanisms that control synapse assembly and function in the nervous system. Knowledge of the regulatory events that control the formation of normal functional connections will greatly enhance our ability to identify, and possibly reverse, the deficits that occur in neurons developing in an abnormal environment due to disease, injury or developmental disorders.